Table 7 Sample preparation procedure for LC-MS/MS analysis
Procedure | Notes | |
|---|---|---|
I. Reduction and alkylation of proteins | ||
1. | Add denaturing buffer to 50 μg protein of protein sample on ice (total volume should be 220 μL). | · Denature protein. |
· Can deal with samples at r.t. after denaturing protein samples. | ||
·Should use low-protein-adsorption 1.5-mL tubes, e.g., SUMITOMO BAKELITE, SUMILON Proteosave SS 1.5 mL tubes, MS-4215 M. | ||
· Do not pipet to prevent adsorption of proteins in pipette tips. | ||
2. | Add same amount of DTT as protein amount (Add 1 μL of 50 μg/μL DTT solution). | · Do not pipet. |
3. | Stir the sample using a tube mixer (e.g., cute mixer CM-1000, EYELA) for 60 min at r.t. | · Reduction of S-S bond. |
4. | Add 2.5-fold IAA of protein amount (Add 2.5 μL of 50 μg/μL IAA solution). | · Do not pipet. |
5. | Stir the sample using a tube mixer for 60 min at r.t. in the dark. | · Protection of –SH residue (alkylation) |
· IAA can be degraded by light, so the sample tubes should be protected from light. | ||
II. Methanol-chloroform precipitation (on ice) | ||
6. | Add 600 μL cold methanol to sample solution. Invert the tube. | · Do not pipet. |
7. | Add 150 μL cold chloroform to sample solution. Invert the tube. | · Do not pipet. |
8. | Immediately after adding 450 μL cold water to sample solution and inverting the tube, centrifuge the sample using swing rotor at 15,000 rpm for 5 min at 4°C. | · Do not pipet. |
9. | Immediately after centrifugation, remove the upper layer (until a floating pellet). | · The floating pellet is protein. |
· Do not take the protein pellet. | ||
10. | Add 450 μL cold methanol to sample solution. Invert the tube gently to wash the protein pellet. | · Do not pipet. |
11. | Centrifuge sample using swing rotor at 15,000 rpm for 5 min at 4°C. | |
12. | Immediately after centrifugation, remove the supernatant. | · Do not take the protein pellet. |
13. | Again centrifuge sample using swing rotor at 15,000 rpm for 1 min at 4°C and remove the supernatant completely. | · Do not take the protein pellet. |
III. Double digestion with LysC and trypsin | ||
14. | Add 9 μL 6 M urea solution, and stir the sample using tube mixer for approximately 10 min at r.t. | · Do not pipet. |
15. | Add 36 μL 0.1 M Tris–HCl buffer (pH 8.5). | · Final concentration of urea is 1.2 M. |
· Do not pipet. | ||
16. | Resuspend protein pellet by intermittent sonication with Branson 2510 sonicator. | · Sonication for 30 seconds followed by a pause for 30 seconds on ice. Repeat this step until the pellet is resuspended. |
17. | Add 1/100-fold LysC of the protein amount (add 1 μL 0.5 μg/μL LysC solution). | · Do not pipet. |
· Gently tap using finger to stir sample solution. | ||
18. | Add 1% ProteaseMax solution (2.5 μL) so that the final concentration is 0.05%. | · Do not pipet. |
· Gently tap using finger to stir sample solution. | ||
19. | Incubate sample at 25°C for 3 h. | |
20. | Add 1/100-fold TPCK-trypsin of the protein amount (Add 1 μL of 0.5 μg/μL TPCK-trypsin solution). | · Do not pipet. |
· Gently tap using finger to stir sample solution. | ||
21. | Incubate sample at 37°C for 16 h. | · Total volume is 49.5 μL. |
IV. LC-MS/MS analysis | ||
22. | Add 7.5 μL of IS peptide mixture. | · The concentration of the IS peptide mixture should be adjusted so that the injected amount of each peptide is 500 fmol for HPLC-API5000 or 100 fmol for HPLC-QTRAP5500. |
· Pipet well in sample solution when adding IS peptide solution, then mix with vortex mixer. | ||
23. | Add 3 μL 50% formic acid in water. | · Acidification. |
· Mix with vortex mixer. | ||
· Total volume is 60.0 μL. | ||
24. | Centrifuge sample at 15,000 rpm for 5 min at 4°C with an angular rotor. | |
25. | Apply 58 μL supernatant to 96-well plates or vials in autosampler. Keep the autosampler at less than 10°C. | |
26. | Inject 40 μL on LC-MS/MS. | · 40 μL includes 33.3 μg peptide sample (50 μg protein × 40 μL/60 μL) and 100 fmol (QTRAP5500) or 500 fmol (API5000) of IS peptides. |